1. Field of the Invention
The present invention relates to a method for the extraction of ATP from a microorganism and, more particularly, to a method for such an extraction that reduces subsequent distortions on assay with luciferase. The method of the invention is particularly useful in the ATP-luciferin-luciferase assay, which is commonly used to monitor microorganism contamination in the food manufacturing industry.
2. Background of the Technology
Many industries have a need for rapid microbial or xe2x80x9cbioburdenxe2x80x9d monitoring. One of the most visible industries with this requirement is the food manufacturing industry. Food manufacturers are typically required, through governmental regulation or through internal operating procedures, to monitor (1) incoming raw materials, (2) in-process manufacturing areas, (3) manufacturing surfaces and/or (4) the food items themselves for contamination with microorganisms. In general, these manufacturers are interested in measuring only viable or living organisms as opposed to non-viable organisms, which do not present a threat to human health.
One method that has gained importance for these applications involves the use of adenosine triphosphate (xe2x80x9cATPxe2x80x9d) bioluminescence (xe2x80x9cATP bioluminescencexe2x80x9d), wherein the firefly luciferin/luciferase system and ATP extracted from a microbial sample are used. If present in a sample, ATP, which is an energy molecule found in all living cells, combines with luciferin in the presence of magnesium ion at approximately neutral pH to form an ATP magnesium-luciferin complex. This combination seems to be driven by charged interactions. The ATP-magnesium-luciferin complex then interacts with the enzyme luciferase in the presence of oxygen to produce light, the intensity of which can be detected using a sensitive light detector. The intensity, measured in relative light units (xe2x80x9cRLUsxe2x80x9d), is directly proportional to the amount of ATP in the sample, and thus can be correlated with the level of microorganisms in the sample. It is well known in the art that only viable or live organisms have measurable levels of ATP. In the case of non-viable organisms, the ATP originally present would have been converted to ADP or AMP by normal biological processes and would be, therefore, unavailable for measurement in the firefly luciferin-luciferase system.
There are several drawbacks associated with prior known ATP-luciferin-luciferase assay methods, including:
1. The enzyme luciferase is labile and, therefore, has a relatively short period of activity in the purified state. Once deterioration of this enzyme occurs, light output (intensity) decreases;
2. The extraction procedures commonly used to liberate ATP from microorganisms in a sample usually involve the use of cationic extractants such as benzalkonium chloride, benzethonium chloride and dodecyl trimethyl ammonium bromide. Cationic extractants are known to have a negative affect on luciferase, presumably by interacting with its active site. Use of cationic extractants, therefore, negatively affects the output of light generally observed during ATP-luciferin-luciferase interaction. See Siro et al., European Journal of Applied Microbiology and Biotechnology; 15:258-64 (1982) and Lundin, Analytical Applications of Bioluminescence and Chemiluminescence, Kricka et al., (eds.), Academic Press, London (1984); and
3. The substances commonly used as sanitizers in sample collection and preparation usually contain harsh substances, eg., bleach, sodium tri-phosphate and quaternary ammonium salts. These substances are also known to have a negative affect on luciferase activity. Similar to the use of cationic extractants, use of such substances during sample collection affects the output of light generally observed during ATP-luciferin-luciferase interaction.
U.S. Pat. No. 5,188,965 discloses a means to remedy drawback (1) above, wherein luciferase is presented in a test device in a dried and, therefore, more stabilized state.
One attempt to remedy drawback (2) above, has been to dilute the cationic extractant prior to use. For example, extraction methods such as that described in the 1980""s literature, which involve the use of, e.g., trichloroacetic acid or dimethyl sulfoxide (DMSO), although effective in extracting ATP from samples, required dilution of the ATP extract prior to light measurement. See Stanley, Methods in Enzymology, 133:14-22 (1986). Unfortunately, this methodology adds steps and time to the overall process and diminishes assay sensitivity.
Attempts to remedy drawback (3) above involve removal of the harsh substance(s) prior to assay. Such attempts, which add steps and time to the overall process, also diminish assay sensitivity.
U.S. Pat. No. 5,004,684 discloses another attempt to alleviate the negative effects of commonly used extractants, as well as to simplify the testing process (i.e., by reducing the number of required steps). According to U.S. Pat. No. 5,004,684, neutral or non-ionic detergents, e.g., polyoxyethylene sorbitan monooleate (Tween 80) and the like, are added to the extraction reaction mixture. The neutral or non-ionic detergents apparently prevent interaction between the cationic detergent and the hydrophobic active site of luciferase (see DeLuca, Purification and Properties of Firefly Luciferase, Methods in Enzymology, 57:3-15; Denburg et al., Arch. Biochem. Biophys., 141:668 (1970)). Although the method disclosed in U.S. Pat. No. 5,004,684 has proven effective, there remains a need in the industry for improved assay sensitivity.
It is an object of the invention to provide a method for extracting ATP from a biological sample.
Another object of the invention is to provide a method for assaying for the presence of ATP in a biological sample.
Yet another object of the invention is to provide a method for detecting the amount of ATP extracted from a biological sample.
A further object of the invention is to provide a method for detecting contamination. The method can be used, for example, for detecting contamination on a surface or in a food product.
Yet a further object of the invention is to provide a reagent, a test device and a test kit for detecting contamination of a sample.
In accordance with the above objects, the method for ATP extraction involves introducing a cationic extractant and anionic substance to extract the ATP. The anionic substance neutralizes a positive charge of the cationic extractant.
The anionic substance is preferably a sulfate ion, more preferably in the form of a magnesium salt, or SDS. The magnesium salt is preferably present in an amount of about 0.0001 xcexcg to about 0.4 xcexcg, and the SDS is preferably present in an amount of about 0.0001 xcexcg to about 0.5 xcexcg.
The method for assaying for the presence of ATP in a biological sample involves introducing a cationic extractant and anionic substance to extract ATP from the biological sample; permitting luciferin and magnesium to react with the extracted ATP to form an ATP-magnesium-luciferin complex; allowing the ATP-magnesium-luciferin complex to interact with luciferase, wherein light is produced; and measuring the intensity of the light, wherein the presence of light corresponds to the presence of ATP.
The method for detecting the amount of ATP extracted from a microorganism involves introducing a cationic extractant and anionic substance to extract the ATP; permitting luciferin and magnesium to react with the extracted ATP to form an ATP-magnesium-luciferin complex; allowing the ATP-magnesium-luciferin complex to interact with luciferase, wherein light is produced; measuring the intensity of the light, wherein the intensity of the light corresponds to the amount of ATP extracted.
The method for detecting contamination in a sample involves extracting ATP from the sample by introducing a cationic extractant and anionic substance to extract the ATP from any microorganisms present therein; permitting luciferin and magnesium to react with the extracted ATP to form an ATP-magnesium-luciferin complex; allowing the ATP-magnesium-luciferin complex to interact with luciferase, wherein light is produced; and measuring the intensity of the light, wherein the intensity of the light corresponds to an amount of ATP extracted, and wherein the amount of ATP extracted corresponds to contamination.
The methods described above may be conducted in the presence of sanitizers commonly used in biological sample collection and preparation.
The test device for performing the above-described method includes a reagent source having the reagents required for the bioluminescence assay contained therein in solid form.
The reagents include a reagent comprising a cationic extractant and an anionic substance. The reagents may further include all substances necessary to carry out the bioluminescence assay, including at least one bioluminescence reagent.
The test kit includes a reagent containing at least a cationic extractant and an anionic substance and, optionally, a bioluminescence reagent. The test kit may include the reagent(s) in the form of a test device.